title: Phage display to identify Nedd8-mimicking peptides as inhibitors of the Nedd8 transfer cascade.
abstract: The Nedd8 activating enzyme (NAE) launches the transfer of the ubiquitin-like protein Nedd8 through an enzymatic cascade to covalently modify a diverse array of proteins, thus regulating their biological functions in the cell. The C-terminal peptide of Nedd8 extends deeply into the active site of NAE and plays an important role in the specific recognition of Nedd8 by NAE. We used phage display to profile C-terminal mutant sequences of Nedd8 that could be recognized by NAE for the activation reaction. We found that NAE can accommodate diverse changes in the Nedd8 C-terminal sequence (⁷¹ LALRGG⁷⁶), including Arg and Ile replacing Leu71, Leu, Ser, and Gln replacing Ala72, and substitutions by bulky aromatic residues at positions 73 and 74. We also observed that short peptides corresponding to the C-terminal sequences of the Nedd8 variants can be activated by NAE to form peptide~NAE thioester conjugates. Once NAE is covalently loaded with these Nedd8-mimicking peptides, it can no longer activate full-length Nedd8 for transfer to the neddylation targets, such as the cullin subunits of cullin-RING E3 ubiquitin ligases (CRLs). We have thus developed a new method to inhibit protein neddylation by Nedd8-mimicking peptides.
authors.affiliation: Department of Chemistry, University of Chicago, 929 E57th Street, Chicago, IL 60637, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
authors.forename: Keya
authors.initials: K
authors.lastname: Zhang
authors.forename: Eric B
authors.initials: EB
authors.lastname: Villhauer
authors.forename: Karan
authors.initials: K
authors.lastname: Bhuripanyo
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kiyokawa
authors.forename: Hermann
authors.initials: H
authors.lastname: Schindelin
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D000075687:NEDD8 Protein; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D014452:Ubiquitins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D019151:Peptide Library; D010455:Peptides; D014452:Ubiquitins
keywords: Nedd8; Nedd8 activating enzymes; enzyme inhibitors; peptides; phage display